LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.
A frog brain kainic acid receptor (KAR) was studied using monoclonal and polyclonal antibodies against the affinity-purified receptor. Immunocytochemistry was done on sections of the frog CNS, and the distribution of immunostaining was compared with the distribution of high- and low-affinity 3H-kainic acid (3H-KA) binding sites determined with in vitro receptor autoradiography. These studies showed (1) similar distributions of high- and low-affinity 3H-KA binding sites, (2) identical patterns of immunostaining with the polyclonal antibodies and 2 monoclonal antibodies, and (3) an antibody binding distribution which closely matched that of 3H-KA binding, suggesting that the antibodies recognize the primary KAR in frog brain. In the frog brain, an anteroposterior gradient of immunostaining was observed, with the telencephalon intensely and uniformly immunoreactive. Other areas intensely immunoreactive included the cerebellum, the infundibulum, the tectal and posterior commissures, and the laminar nucleus of the torus semicircularis. The optic tectum showed selective staining of the plexiform layers 3 and 5–7. The pattern of staining was punctate and appeared to be associated with nerve fibers, among them dendritic arborizations. Electron microscopic observations showed staining at the cytoplasmic side of postsynaptic membranes. Extra-synaptic staining was observed as patches on the surface of unmyelinated nerve processes.
The regulation of mucin secretion by SW1116 human colon carcinoma cells has been studied using monoclonal antibody 19-9, which has previously been used to detect mucin in the serum of cancer and cystic fibrosis patients. We found that SW1116 cells constitutively secrete considerable amounts of mucin as the predominant glycoprotein. The secretion of mucin by these cells is independent of cyclic AMP levels, but can be further stimulated by the Ca2+ ionophore A23187. However, arachidonic acid and its metabolites inhibit mucin secretion. Electron microscope studies reveal that the mucin is located near the plasma membrane as well as in vesicular and lysosome-like structures. However, the secretion pathway of mucin is different than that of the lysosomal contents, since arachidonic acid, while inhibiting mucin secretion, actually activates the secretion of the lysosomal enzyme beta-glucuronidase. We suggest that the mechanism of mucin secretion by SW1116 cells occurs by a pathway different from common exocytosis, and possibly by more than one pathway. The response of mucin secretion by SW1116 cells to common secretagogues resembles that of epithelial cells obtained from cystic fibrosis patients. Thus SW1116 cells are an especially interesting system for studying processes related to pathological states associated with excessive constitutive secretion of mucin.
Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo.To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology.Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control islets that are principally associated with cell division and DNA repair. The latter genes have not specifically been associated with islet physiology in the past. By contrast, Anx7(+/-) mouse islets exhibit a greatly reduced ability to discriminate genomically between fed and fasted states for all classes of identified genes. Many of the validated genes are specific to islets in comparison to liver tissue examined. Real-time quantitative RT-PCR analysis of islets from Anx7 heterozygous mice and littermate controls revealed remarkable down-regulation in PTEN, Glut-2, PDX-1, IGF-1, and Neuro D1 expression, but not in liver.We conclude that reduced gene dosage in the Anx7(+/-) islet, with concomitant loss of ITPR3 expression and consequent defects in Ca(2+) signaling, may substantially contribute to the mechanism of the loss of genomic discrimination, in vivo, between the fed and fasted states. We believe that the requirement for complete Anx7 gene dosage and IPTR3 expression in islets of Langerhans will prove to be of fundamental importance for understanding the mechanism of nutritional sensing in health and disease.
